Analysis of the sequence features contributing to centromere organisation and CENP-A positioning and incorporation

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Abstract

Centromere identity is integral for proper kinetochore formation and chromosome
segregation. In most species chromosomes have a centromere at a defined locus
that is propagated across generations. The histone H3 variant CENP-A acts as an
epigenetic mark for centromere identity in most species studied. CENP-A is absent
from the inactivated centromere on dicentric chromosomes and present at
neocentromeres that form on non-centromeric sequences. Thus, the canonical
centromere sequence is neither necessary nor sufficient for centromere function.
Nevertheless, centromeres are generally associated with particular sequences.
Understanding the organisation of centromeric sequence features will provide
insight into centromere function and identity.
In this study I use the fission yeast Schizosaccharomyces pombe model system to
address the relationship between CENP-ACnp1 and centromeric sequence features.
These analyses reveal that CENP-ACnp1 nucleosomes are highly positioned within
the central domain by large asymmetric AT-rich gaps. The same sequence features
underlying CENP-ACnp1 positioning are conserved in the related species S.
octosporus, but are not found at neocentromeres, suggesting that they are important
but non-essential for centromere function. CENP-ACnp1 over-expression leads to
ectopic CENP-ACnp1 incorporation primarily at sites associated with heterochromatin,
including the sites where stable neocentromeres form. Ectopic CENP-ACnp1 also
occupies additional sites within the central domain that are not occupied in cells with
wild-type CENP-ACnp1 levels. In wild-type cells CENP-ACnp1 occupied sites are likely
also occupied by H3 nucleosomes or the CENP-T/W/S/X nucleosome-like complex
in a mixed population. Several candidate proteins were investigated to determine a
protein residing in the large gaps between CENP-ACnp1 nucleosomes could be
identified. No proteins could be localised to the AT-rich gaps between CENP-ACnp1
nucleosomes, but the origin recognition complex in a promising candidate.
The results presented in this thesis demonstrate that nucleosomes within the fission
yeast centromere central domain are highly positioned by sequence features in a
conserved manner. This positioning also allows for another complex, possibly the
origin recognition complex, to bind to DNA. Nucleosome positioning, DNA
replication, and transcription could individually and collectively influence CENP-ACnp1
assembly and centromere function. Further experiments in fission yeast will
continue to provide insight into the general properties of centromere function and
identity.